BACKGROUND

Multicellular tumor spheroids (MCTS) are advanced cell culture systems for assessing the impact of combinatorial radio(chemo)therapy as they exhibit therapeutically relevant in vivo-like characteristics from 3-dimensional cell-cell and cell-matrix interactions to radial pathophysiological gradients. State-of-the-art assays quantify long-term curative endpoints based on collected brightfield image time series from large treated spheroid populations per irradiation dose and treatment arm. This analyses require laborious spheroid segmentation of up to 100,000 images per treatment arm to extract relevant structural information from the images (e.g., diameter, area, volume, and circularity). While several image analysis algorithms are available for spheroid segmentation, they all focus on compact MCTS with a clearly distinguishable outer rim throughout growth. However, they often fail for the common case of treated MCTS, which may partly be detached and destroyed and are usually obscured by dead cell debris.

RESULTS

To address these issues, we successfully train 2 fully convolutional networks, UNet and HRNet, and optimize their hyperparameters to develop an automatic segmentation for both untreated and treated MCTS. We extensively test the automatic segmentation on larger, independent datasets and observe high accuracy for most images with Jaccard indices around 90%. For cases with lower accuracy, we demonstrate that the deviation is comparable to the interobserver variability. We also test against previously published datasets and spheroid segmentations.

CONCLUSIONS

The developed automatic segmentation can not only be used directly but also integrated into existing spheroid analysis pipelines and tools. This facilitates the analysis of 3-dimensional spheroid assay experiments and contributes to the reproducibility and standardization of this preclinical in vitro model.